Reforestation, the controlled regeneration of forests, has become an integral part of forest management in order to secure a renewable and sustainable source of raw material for production of paper and other wood-related products. Forest trees can be regenerated by either sexual or asexual propagation. Sexual reproduction of seedlings for reforestation has traditionally been the most important means of propagation, especially with coniferous species.
Tree improvement programs with economically important conifers (e.g., Pinus, Picea, and Pseudotsuga species) have applied genetic principles of selection and breeding to achieve genetic gain. Based on the results of progeny tests, superior maternal trees are selected and used in "seed orchards" for mass production of genetically improved seed. The genetic gain in such an open-pollinated sexual propagation strategy is, however, limited by the breeder's inability to control the paternal parent. Further gains can be achieved by control-pollination of the maternal tree with pollen from individual trees whose progeny have also demonstrated superior growth characteristics. Yet sexual propagation results in a "family" of seeds comprised of many different genetic combinations (known as siblings), even though both parents of each sibling seed are the same. As not all genotype combinations are favorable, the potential genetic gain is reduced due to this genetic variation among sibling seeds.
In addition to these genetic limitations, large-scale production of control pollinated seeds is expensive. These economic and biological limitations on large-scale seed production have caused considerable interest to develop in the industry for applying asexual methods to propagate economically important conifers.
The use of asexual propagation permits one to apply what is known as a very high selection intensity (that is, propagate only progeny showing a very high genetic gain potential). These highly desirable progeny have unique genetic combinations that result in superior growth and performance characteristics. Thus, with asexual propagation it is possible to multiply genetically select individuals while avoiding a concomitant reduction of genetic gain due to within family variation.
Asexual propagation of trees can be accomplished by methods of grafting, vegetative propagation, and micropropagation. Grafting, widely used to propagate select individuals in limited quantities for seed orchard establishment, is not applicable to large-scale production for reforestation. Vegetative propagation by rooting of cuttings and micropropagation by somatic embryogenesis currently hold the most potential for reforestation of coniferous trees. Although vegetative propagation by rooted cuttings can be achieved in many coniferous species, large-scale production via this method is extremely costly due to difficulties in automating and mechanizing the process. This propagation method is further limited by the fact that the rooting potential of stock plants decrease with time, making it difficult to serially propagate from select genotypes over extended periods of time.
Micropropagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from small pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Both vegetative propagation and micropropagation have the potential to capture all genetic gain of highly desirable genotypes. However, unlike conventional vegetative propagation methods, somatic embryogenesis is amenable to automation and mechanization, making it highly desirable for large-scale production of planting stock for reforestation. In addition, somatic embryogenic cultures can easily be preserved in liquid nitrogen. Having a long-term cryogenic preservation system offers immense advantages over other vegetative propagation systems which attempt to maintain the juvenility of stock plants.
The current invention specifically relates to the development of an improved cell and tissue culture system for micropropagation of conifers by somatic embryogenesis. It was not until 1985 that somatic embryogenesis was discovered in conifers (Hakman et al. 1985) and the first viable plantlets were regenerated from conifer somatic embryos (Hakman and von Arnold 1985). Since 1985 conifer tissue culture workers throughout the world have pursued the development of somatic embryogenesis systems capable of regenerating plants. The goal of much of this work is to develop conifer somatic embryogenesis as an efficient micropropagation system for producing clonal planting stock enmasse. In addition, the embryogenic micropropagation system interfaces very well with genetic engineering techniques for production of transgenic clonal planting stock of conifers.
The two most economically important conifer genera are Picea (spruce) and Pinus (pine). There are about 30 species of Picea, largely restricted to cooler regions of the northern hemisphere, of which seven species are native to North America. Pinus is the largest and most important genus of conifers, having approximately 95 species scattered over the northern hemisphere. Of these 95 species, 36 are native to North America. (Preston 1989) .
Those working in conifer somatic embryogenesis have found that there is a striking difference between Picea conifers and Pinus conifers as to the ease with which somatic embryogenesis can be induced and plants regenerated (Tautorus et al. 1991). Indeed, if one evaluates the success of somatic embryogenesis in conifers among species of these two important genera, it is clear that much more success has been achieved with Picea than with Pinus. It is also striking how consistent the success on developing somatic embryogenic systems has been among several Picea species, whereas the recalcitrance of Pinus has been equally consistent across several species.
Progress in somatic embryogenesis can in part be evaluated by the level of success in three important steps of the process: (1) initiation of embryogenic cultures, (2) production of fully developed somatic embryos, and (3) establishment of plants under field conditions. Among Picea species embryogenic culture initiation frequencies are relatively high; as high as 95% from immature zygotic embryos, and as high as 55% from mature zygotic embryos harvested from fully developed, dry seeds (Tautorus et al. 1991). There are numerous reports of production of fully developed somatic embryos among Picea species, and several reports of establishment and growth of Picea plants in soil. Researchers at the British Columbia Research Corporation have reported on establishment of interior spruce (a mixture of Picea glauca and Picea englemannii) plants under nursery conditions. For example, Webster et al. (1990) reported over 80% survival and establishment in nursery conditions of interior spruce plants for most of 71 genotypes tested. Grossnickle et al. (1992) reported the establishment of 40% of 2000 interior spruce plants in nursery conditions. The plants were derived from 15 different genotypes. Researchers at the Weyerhaeuser Corporation have reported similar success with Norway spruce (Picea abies); over 3000 plants from 17 genotypes have been established in the field (Gupta et al. 1992). Similar success was also reported with Douglas-fir (Pseudotsuga menziesii); over 2000 plants from 6 genotypes have been established in soil under greenhouse conditions. Thus, conifer somatic embryogenesis workers utilizing Picea species (and commercially important Douglas-fir) have been successful in developing culture initiation and regeneration systems that enable relatively routine production of plants capable of transfer to field conditions. The rapid successes in Picea somatic embryogenesis had lead to considerable optimism among researchers that commercial utilization of conifer somatic embryogenesis for production of clonal planting stock of Pinus conifers would be readily achievable.
However, the progress achieved with somatic embryogenesis in Pinus species to date has been much less encouraging than that achieved with Picea species. First and foremost in difficulty is the recalcitrance of Pinus species for initiation of embryogenic cultures. For example, initiation frequencies of about 1 to 5% are routinely cited by those working with Pinus species (Gupta and Durzan 1987, Becwar et al. 1988, Jain et al. 1989, Becwar et al. 1990). The single report claiming a 54% initiation rate from immature zygotic embryos of Pinus strobus (Finer et al. 1989) has yet to be repeated or duplicated by others working with this species (Michler et al. 1991). Secondly, it is extremely difficult to obtain reliable development of Pinus somatic embryos to the fully developed (cotyledonary) stage. In addition, subsequent production of plantlets has been extremely limited in Pinus species. Tautorus et al. (1992) cited only 3 of 7 reports which indicated plantlets were obtained via somatic embryogenesis in Pinus species. (In contrast, 30 of 43 reports with Picea species reported obtaining plantlets via somatic embryogenesis.) Unlike the reports with Picea species where several systems have shown potential for plantlet production on relatively large scales, the reports of plantlet production from Pinus species have yielded few plants. To our knowledge there is only one report of successful establishment of Pinus somatic embryos in soil (Gupta and Durzan 1987). The authors of this report have had limited success in obtaining Pinus taeda plants . . . indeed, only one culture genotype was taken to the plantlet stage and only one plant was transferred to soil (see Pullman and Gupta 1991). To date the only published report of higher numbers of germination of Pinus somatic embryos is for Pinus caribaea, where 34 of 69 (49%) germinated (Laine and David 1990). However, the authors did not report establishment of these plants in field conditions. Thus, for Pinus species all three integral parts of the somatic embryogenesis process have not progressed to the stages currently achieved with Picea.
Having a low initiation frequency can severely limit the potential applications of somatic embryogenesis in Pinus species for large scale production of genetically improved conifers for the following reason. Skilled artisans in the field of conifer tissue culture recognize that the use of embryogenic cultures derived from juvenile explants (e.g., zygotic embryos derived from seed) necessitate that the resulting regenerated plants be field tested prior to large scale production. Only selected genotypes which show the potential for producing significant genetic gain in such a field test will subsequently be propagated by somatic embryogenesis. Therefore it will be necessary to screen numerous genotypes from desirable parents, initiate embryogenic cultures, cryopreserve each genetically different culture, regenerate plants from each genetically different culture, field test plants from each genotype, and choose select genotypes for large scale production via somatic embryogenesis. Low culture initiation frequencies pose severe limitations for implementing this strategy. Indeed, an unbeknownst selection process may occur when low initiation frequencies exclude a majority of the genotypes. In the case of Pinus species where initiation frequencies are very low (e.g., 1 to 5%) one could be selecting for embryogenic potential, but selecting against improved growth potential (which may be in the 95 to 99% of the genotypes eliminated as non-embryogenic). The potential problem of eliminating desirable genotypes is exacerbated by the exceedingly low initiation frequencies among Pinus species. By contrast, with Picea species where initiation frequencies are much higher (approaching 100% from immature zygotic embryos of some Picea species) it is much less likely that one will eliminate by selection those genotypes which have superior growth potential.
One component of an efficient somatic embryogenesis regeneration system is the culture medium. Semi-solid culture media are routinely used during the culture initiation, the culture maintenance, and the embryo development phases. The culture medium is generally composed of six groups of ingredients: inorganic nutrients, vitamins, organic supplements, a carbon source, phytohormone(s), and a gelling agent for semi-solid media. The two gelling agents usually employed for conifer somatic embryogenesis are agar and gellan gum, with agar being most commonly used.
Gelling agent concentration and type are known to influence growth responses of certain non-coniferous plant tissue cultures, but the effects of gelling agent concentration are varied and complex among different plant species and plant tissue types. For example, in a study working with rose (Rosa hybrida) tissue cultures Ghashghaie et al. (1991) found that increasing the availability of water by lowering a medium's agar concentration increased shoot elongation, yet did not improve shoot multiplication. Etienee et al. (1991) showed that culturing rubber tree (Hevea brasiliensis) explants on cellulose blocks in liquid medium increased embryogenic tissue initiation in comparison to culturing on the same medium gelled with a standard level of 2 grams of GELRITE.RTM. (gellan gum manufactured by Merck, Inc.) per liter of medium (grams/liter or g/l). They suggested the increased initiation was due to increased water availability of the liquid medium relative to the gelled medium. But, they did not determine if culturing explants on medium gelled with low levels of GELRITE (e.g, 1 g/l) similarly increased initiation. In another study utilizing sugarbeet (Beta vulgaris) leaf discs, Owens and Wozniak (1991) obtained more somatic embryos and shoots from leaf discs cultured on low levels of gelling agent. However, their results were obtained from sugarbeet explants cultured on a filter-paper overlay. The study did not directly evaluate how varying gelling agent concentration effected somatic embryo production from sugarbeet explants cultured directly on the culture medium surface.
Those working in the field of conifer somatic embryogenesis have mainly emphasized medium components other than the gelling agent in attempts to improve culture initiation or development of somatic embryos (Tautorus et al. 1991). Only four reports have examined the effect of gelling agents on conifer somatic embryogenesis (von Arnold 1987, Klimaszewska 1989, Harry and Thorpe 1991, and Tremblay and Tremblay 1991). In her study von Arnold (1987) compared agar to GELRITE and found no difference between the two gelling agents for initiation of embryogenic tissue from mature zygotic embryos of Picea abies. The study did not test media gelled with levels of agar and GELRITE below 7 and 2 g/l, respectively. Klimaszewska (1989) compared the effect of agar versus GELRITE on proliferation and growth of Larix embryogenic cultures. Cultures initiated on medium gelled with 7 g/l of agar proliferated and grew best when transferred to medium gelled with 4 g/l of GELRITE. Although her study did not examine the effects of low levels of gelling agents on the culture initiation step, Klimaszewska did conclude that it was extremely difficult to maintain high quality cultures on a medium containing a low level of GELRITE (1 g/l)--thereby teaching away from the use of low levels of gelling agents. Harry and Thorpe (1991) tested the effect of agar and GELRITE concentration on initiation of Picea rubens embryogenic tissue, but did not test levels below 6 and 2 g/l, respectively. Tremblay and Tremblay (1991) examined the effect of gelling agents on the development of Picea abies and Picea rubens somatic embryos. They found that GELRITE was superior to agar, in that 3 to 5 times more somatic embryos developed on medium gelled with GELRITE than with agar. However, concentrations of agar and GELRITE below 7 and 2 g/l, respectively, were not tested.
Researchers in conifer somatic embryogenesis have commonly employed the same levels of gelling agents typically used in other plant cell and tissue culture research. These traditional gelling agent levels are 6.0 to 9.0 grams of agar per liter of medium, 2.0 to 4.0 g/l of gellan gum (or GELRITE), 6.0 to 10.0 g/l of agarose (a purified form of agar), and 3.5 to 5.0 g/l of AGARGEL.RTM. (an agar/gellan gum mixture manufactured by Sigma Chemical Co.). Although Hakman et al. (1985) employed an agar level of 5 g/l in a study to induce somatic embryogenic cultures of Picea abies, no suggestion was made by the authors of any significance or advantage to using this level. Indeed, in subsequent studies these authors exclusively used higher levels of GELRITE (3 to 4 g/l) (Hakman and von Arnold 1985, von Arnold and Hakman 1988). To our knowledge, no one heretofore has explored the efficacy of using low levels of gelling agents for culture initiation of somatic embryogenesis among conifers.
The implementation of somatic embryogenesis in Pinus species for production of clonal planting stock is also severely limited by the lack of a reproducible multi-step regeneration system. Very few laboratories working with Pinus have effectively produced embryogenic cultures or even produced cotyledonary stage somatic embryos. Even fewer workers have regenerated Pinus plants by somatic embryogenesis (Tautorus et al. 1991). In the cases where plants have been regenerated from Pinus embryogenic cultures, both the number of responsive culture genotypes and the number of plants obtained have been very low.
The present invention is a multi-step somatic embryo regeneration method that is applicable to Pinus species and has demonstrated potential to regenerate plants from a diverse range of culture genotypes. The invention method also improves the embryogenic culture initiation frequency. This in itself is highly significant because it ensures that more embryogenic cultures survive to the culture maintenance phase, thereby allowing more genotypes to be subsequently available for field testing and production of clonal planting stock.
In U.S. Pat. No. 4,957,866, Gupta et al. teach a process for reproducing coniferous plants (i.e. Pinus taeda) via somatic embryogenesis. Direct comparisons were performed between the patented process and the method taught in the present invention (see Examples 5 and 6 below). The results contained in Example 5 clearly showed that the current invention method provides a significant improvement in culture initiation when compared to the Gupta et al. process. (As noted above, it is vitally important to improve the culture initiation method practiced with Pinus in order to assure that more embryogenic culture genotypes are initiated and available for use in subsequent steps of the regeneration method.) In Example 6 the process of increasing the predevelopment medium osmotic potential disclosed in the Gupta et al. patent was compared to the method taught in the current invention. There the results achieved across several culture genotypes were at least equivalent, and in most cases far better, using the method of the current invention.
In U.S. Pat. No. 5,034,326, Pullman and Gupta teach a process for reproducing coniferous plants (i.e. Pinus taeda) via somatic embryogenesis which involves using activated carbon and high levels of abscisic acid in the embryo development medium. In Example 6 the use of high levels of abscisic acid and activated carbon in embryo development medium as disclosed by the Pullman and Gupta patent was compared to the method taught in the current invention. This comparison study found the method taught in the current invention to be very effective while, in contrast, the patented process was found to be ineffective.
In U.S. Pat. No. 5,036,007, Gupta and Pullman teach a process for reproducing coniferous plants via somatic embryogenesis which involves using abscisic acid and osmotic potential variation of the culture medium. In addition to utilizing high levels of abscisic acid in combination with activated carbon, they also teach using a subsequent embryo development medium having very high osmolality levels (preferably in the range of about 450 mM/kg). The current invention differs significantly from both of the above patented processes (U.S. Pat. Nos. 5,034,326 and 5,036,007). First, in the current invention activated carbon is not used in combination with abscisic acid. Second, the current invention does not require the embryo development medium to have the high osmolality levels as taught by Gupta and Pullman (1991).
Several species of pine indigenous to the Southeastern United States are closely related and hybridize naturally. Taxonomically this group of pines is referred to as Southern yellow pines and includes Pinus taeda, P. serotina, P. palustris and P. elliottii (Preston 1989).
In addition to their taxonomical similarity, the above pine species have responded alike in studies on somatic embryogenesis attempts. For example, all previous reports of somatic embryogenesis with the above species have found the same stage, very early precotyledonary zygotic embryos, to be optimum for embryogenic culture initiation (Becwar et al., 1989 & 1990, Jain et al. 1989, Nagmani et al. 1993). Initiation frequencies were similarly low (about 1 to 5%) among these species. In all of the above reports the researchers were unsuccessful in obtaining somatic embryo development beyond the very early precotyledonary stage. Thus, there is strong evidence for similarity of the biological response of these species to somatic embryogenesis and that completion of the somatic embryogenesis process is very difficult to achieve in any of these closely related southern pines.
Therefore, an object of the present invention is to provide a method for mass producing clones of Pinus conifers, particularly Southern yellow pine conifers, by the process of somatic embryogenesis.
Another object of the present invention is an improved embryogenic culture initiation method for Pinus conifers.
A further object of the present invention is to provide a multistage regeneration protocol which can be utilized effectively on Pinus conifers to produce large quantities of plants for field planting.
Another object of the present invention is to provide a progression of steps which, in combination, enable one to complete the somatic embryo regeneration method on a number of diverse genotypes of Pinus taeda and other Pinus species.
In addition, it is the object of the present invention to provide a progression of steps which, in combination, enable one to complete the somatic embryo regeneration method on a number of diverse genotypes of Pinus hybrids (e.g., Pinus taeda.times.Pinus rigida).